A specialized function of white adipose tissue is to store neutral lipids within protein-coated droplets. This lipid storage function provides both an energy reservoir and a site to sequester excess lipids that otherwise would have toxic effects on tissues such as muscle, blood vessels, liver, and pancreas, thereby increasing the risk for diabetes mellitus, heart disease, and nonalcoholic fatty liver disease. How newly synthesized triacylglycerol is packaged and inserted into lipid storage droplets is not understood. Also, little is known about the identity and function of the proteins that coat the lipid droplet. The overall goal of this research plan is to discover the mechanisms by which lipids traffic intracellularly to their site of storage within the adipocyte. Understanding such mechanisms may illuminate what goes wrong in conditions of excessive lipid storage (obesity) and inefficient partitioning of lipid into adipocytes (lipodystrophy /lipoatrophy). The preliminary data reveal a process of lipid droplet maturation in which the adipocyte protein S3-12 coats newly formed lipid droplets and promotes triacylglycerol accumulation. We hypothesize that S3- 12 promotes efficient packaging of newly synthesized triacylglycerol into storage droplets by coating nascent lipid droplets and mediating their incorporation into perilipin-coated storage droplets. According to this model, S3-12 serves to channel triacylglycerol into the storage rather than hydrolytic pathway. Aim 1 seeks to define changes in the adipocyte and lipid droplet proteomes during active triacylglycerol packaging. Aim 2 seeks to document in live cells the process of lipid droplet biogenesis and to determine how S3-12 promotes triacylglycerol accumulation and lipid droplet organization. The structural features of S3-12 that mediate its targeting to nascent lipid droplets and its function will be mapped by mutational analysis. Finally, Aim 3 investigates whether global overexpression of S3-12 in the mouse will promote triacylglycerol accumulation in adipocytes and protect against lipotoxicity in other tissues by sequestration of triacylglycerol within storage droplets, thereby limiting lipolysis and reducing the production of toxic lipid metabolites. By these studies, fundamental mechanisms of intracellular lipid trafficking should be revealed.